It is generally agreed that the normal endothelial lining of blood vessels is highly important in rendering the luminal surface of the vascular system non-thrombogenic. This cell type performs this function in part by providing a barrier between the coagulant triggers of the underlying tissue and the procoagulants of circulating blood. Recent cell culture studies have provided evidence that the endothelial cell also synthesizes and secretes many hemostatic factors including plasminogen activator. Presumably, the release of such biologically important substances should be subject to precise regulatory control. Imbalances in this physiological process could thereby contribute to the development of thrombosis. The observation that impaired release of plasminogen activator correlates with an increased tendency for thrombotic episodes, has led us to investigate the vascular derived plasminogen activator and the physiological factors governing its release in animal model. The proposed research will focus on 1) the isolation and purification of a canine vascular-derived plasminogen activator by several appraoches, including endothelial cell culture; 2) a study of the mechanism of plasminogen activation by the activator and its interaction with certain components in plasma and with those produced by the vessel wall; 3) the production of monospecific antiserum to the purified activator for use in a) quantitating the distribution of this activator in this species (tissues, blood vessels, circulating plasma) and b) in vivo studies of the physiological factors which affect its release into the circulation; 4) beginning initial studies on the influence of aging on these processes; and 5) determining the relevance of these findings to the human fibrinolytic system. The methods employed will emphasize protein purifications, enzymatic and immunological assays, gel electrophoresis, immunochemistry, cell culture and isotopic labeling procedures. The integration of both physiological and biochemical results should permit us to construct a model of the in vivo fibrinolytic activation process as it occurs at the blood-vessel wall interface. Furthermore, the findings should have clinical importance in regard to thrombolytic therapy and provide insights into how the fibrinolytic system functions in normal as well as pathological situations.